CN101270334B - Isolated culture method for anaerobic microorganism - Google Patents

Abstract

The invention relates to a method for isolating and cultivating anaerobic microorganisms. The method avoids the contact of target microorganisms with oxygen during the whole operation process, and the operation cost is relatively low. It is especially suitable for those whose growth temperature is higher than 50°C and which have strict requirements on anaerobic environment. Difficult to separate high temperature anaerobic bacteria, strict anaerobic bacteria. This method only needs to use the anaerobic workstation for part of the operation of anaerobic bacteria isolation, and can use an ordinary incubator for cultivation, without continuing to provide an anaerobic environment, avoiding that an anaerobic workstation can only be used for one kind of growth during isolation and cultivation This method is also applicable to bacteria whose culture temperature is higher than the upper limit of the anaerobic workstation.

Description

A method for isolating and cultivating anaerobic microorganisms

field of invention

The present invention relates to a method for separating anaerobic microorganisms, in particular to an indispensable new method for extremophile physiological ecology research, natural ecology research, genetic engineering research, life science research, pathology research, marine geological research and environmental pollution control. method.

Background technique

In natural ecosystems, microorganisms play a very important role. In pollution control, microorganisms can decompose many organic or inorganic pollutants that cannot be removed by other methods, and the cost is low; in geological research, the kinship of specific types of extremophiles in different regions may mark the previous location of these regions relation. However, microorganisms are often based on flora as a unit, and a variety of microorganisms live together. Therefore, the isolation and pure culture of microorganisms is a problem that must be solved first in the study of microbial physiology and biochemistry. The discovery of a new microbial strain often leads to significant progress in microbiology, and may also lead to a new interdisciplinary point: such as the treatment of nitrate-reducing bacteria and nitrate wastewater, Helicobacter pylori and the treatment of gastric cancer, etc. Among them, microorganisms in extreme environments often play an important role in environmental, energy, and geological research, which can provide researchers with many new ideas and methods. However, because the environmental conditions for their cultivation are difficult to simulate under conventional conditions, it is often difficult to purify and cultivate microorganisms in extreme environments, especially when several extreme conditions are required at the same time, such as an extreme anaerobic environment and a high temperature environment.

There are many methods and patents for the isolation and purification of microorganisms, such as a disposable microorganism isolation diagnosis and identification medium (patent number: 200520003682.8), a microorganism isolation and cultivation method (patent number: 94103474.7), etc., but most of these isolation methods are For isolating aerobic or facultative anaerobic bacteria.

Anaerobic bacteria are divided into non-strict anaerobic bacteria and strict anaerobic bacteria: bacteria that will cause growth inhibition when exposed to oxygen are non-strict anaerobic bacteria, and bacteria that cause bacterial death when exposed to oxygen are called strict anaerobic bacteria. Different anaerobic bacteria have different tolerance to oxygen, and the oxygen tolerance time is generally used as an indicator. During the oxygen resistance time, the bacteria will not die, and the oxygen resistance time of the bacteria varies greatly, ranging from a few seconds to tens of hours. For bacteria with a very short oxygen resistance time, because it is easy to cause bacterial death due to contact with oxygen during the separation operation, it is often difficult for people to separate them, and the nucleic acid fragments can only be detected by the probe method, and pure bacteria cannot be obtained. cultivate bacteria. Most of the bacteria with short oxygen resistance time live in extreme environments, such as in the deep sea, due to the huge water pressure, the bacteria will not come into contact with oxygen. Microorganisms in extreme environments are often a new species, which is of great significance to scientific research; and because samples under extreme conditions are difficult to obtain, microorganisms isolated from extreme conditions are very precious. However, this strict requirement for anaerobic conditions creates a great obstacle for isolating anaerobic bacteria. If the pure culture of bacteria is not obtained, it is difficult for people to study the metabolic mechanism of the anaerobic bacteria under natural conditions, and further research on anaerobic bacteria is restricted. In the search for anaerobic bacteria, many bacteria are called "uncultured bacteria". This shows that for anaerobic bacteria, the existing separation technology still has great limitations.

The main methods for isolating anaerobic bacteria have been reported so far:

1) Using an anaerobic workstation to isolate and cultivate bacteria can ensure that the target bacteria are isolated from oxygen during the whole process of isolation. However, the upper limit of the culture temperature generally set in the current anaerobic workstation is 50 ° C, which is difficult to give thermophilic anaerobic bacteria. Aerobic bacteria provide sufficient culture temperature. Moreover, even if it is desired to isolate and cultivate bacteria in a growth environment below 50°C, one anaerobic workstation can only set one cultivation temperature, and to isolate and cultivate microorganisms at different temperatures requires multiple anaerobic workstations, which greatly increases the cost of the experiment. At the same time, it is difficult to provide a strict aseptic environment in the anaerobic workstation, and it is easy to contaminate the petri dishes with each other during cultivation.

2) Using anaerobic tank, anaerobic bag and rolling tube to separate anaerobic microorganisms can provide bacteria with a culture temperature above 50°C, but the whole operation process is exposed to the air, and the target anaerobic bacteria will come into contact with oxygen, resulting in the growth of the target bacteria. body death.

3) The document "An Improved Method for Isolating and Cultivating Sulphate-Reducing Bacteria" (Wan Haiqing, Journal of Applied and Environmental Biology, 2003, 9(5): 561-562) introduces the separation of anaerobic microorganisms by the stacked dish interlayer method, However, in this method, during the coating operation, the bacterium will be exposed to the air in a short period of time. If the oxygen resistance time of the pre-isolated bacteria is less, it will cause the death of the target bacteria.

4) The document "Evaluation of a new anaerobic culture dish-OxyplateTM" (Zhao Hu et al., Shanghai Journal of Medical Laboratory, 2002, 06) introduces the method of utilizing the OxyplateTM culture dish to separate anaerobic bacteria. During the coating operation, the bacteria are exposed to the air for a short time, resulting in the death of the bacteria.

5) A petri dish suitable for anaerobic culture is patented (patent number: 200420074957.2). This patent is aimed at the discovery and cultivation of anaerobic pathogenic bacteria in medical grass-roots institutions. Pathogenic bacteria often live in a non-strictly anaerobic environment In the process, using this culture dish to isolate bacteria with a short oxygen resistance time may cause the death of the bacteria.

Contents of the invention

The purpose of the present invention is to provide a simple and accurate method for isolating high-temperature anaerobic bacteria, which can avoid the contact of target anaerobic bacteria with air during the whole operation, and can separate high-temperature anaerobic bacteria with a culture temperature above 50°C. bacteria.

Concrete solution of the present invention is:

1) Sterilize the agar medium containing the indicator and the glass tube with a sealed cover;

2) Put the unsolidified agar medium, the natural substance containing the target anaerobic bacteria, and the glass tube with a sealed cover into the sample chamber of the anaerobic workstation, repeatedly pump air into the sample chamber three times, and flush nitrogen ( or hydrogen-nitrogen mixture) for routine operation;

3) After mixing the natural substances containing the target microorganisms and the unsolidified agar medium evenly, divide them into glass tubes, and tighten the sealing caps;

4) Take out the glass tube from the anaerobic workstation, place it in the incubator, set the cultivation temperature, and cultivate for a certain period of time;

5) When there is a positive reaction in the glass tube (consistent with the positive reaction of the added indicator), take out the target bacteria in the anaerobic operation station.

The advantages of the present invention are as follows:

1. The separation speed is fast, the time from the enrichment solution to the separation of the bacteria is within one week, and the purity is very high;

2. The operating cost to achieve the same separation effect is lower than that of other high temperature anaerobic bacteria separation methods;

3. Easy to operate;

4. It can isolate high-temperature anaerobic bacteria that are difficult to obtain by ordinary methods.

Description of drawings

Figure 1 The anaerobic tube for cultivating high temperature anaerobic bacteria and the copper wire used for digging agar

1. Copper wire used when digging agar blocks

2. Anaerobic Tubes for Cultivating Thermoanaerobic Bacteria

Figure 2 Schematic diagram of anaerobic workstation

1. Gas system

2. Airtight chamber (for sending samples)

3. Sample

4. The channel connecting the airtight room and the operating room

5. Operating room

Detailed ways

Take natural substances containing target anaerobic microorganisms (hydrothermal vent sediment, bottom surface water, anaerobic sludge), enrich them with a suitable liquid medium, and mix the enriched liquid with uncoagulated solids in an anaerobic workstation. The medium is mixed, divided into anaerobic tubes, taken out from the anaerobic workstation, and cultured under different temperature conditions, and the target strain can be isolated.

Example 1

1. Enrich 2.5 g of the bottom mud of the Pacific Ocean deep-sea hydrothermal vent containing the target bacteria with the liquid medium added with ferrous sulfate heptahydrate (0.0001g/L) indicator.

2. Under aerobic conditions, use ethanol (concentration: 0.2mL/L) as carbon source, ferrous sulfate heptahydrate (0.006g/L) as indicator, agar concentration as 1.8g/L, prepare medium 50mL, Place in a Erlenmeyer flask with a cotton stopper, and sterilize under high temperature and high pressure (121° C., 15 minutes).

3. Before the culture medium is solidified, place the Erlenmeyer flask and the enrichment solution in the sample chamber of the anaerobic workstation, pump nitrogen and fill it with nitrogen repeatedly three times, and remove the air in the culture medium.

4. In the operation box of the anaerobic workstation, add 3mL of the enriched supernatant to the uncoagulated medium, shake well and distribute it into anaerobic tubes (or transparent glass with well-tight screw plugs) tube).

5. Take out the anaerobic tube, put it into an ordinary incubator, and adjust the temperature to 90°C. After 60 hours, it can be observed that there are small black spots (color development phenomenon that reacts with the indicator) inside the medium, and continue to cultivate for 2 Every day, there will be small black spots of different sizes and different times, which are marked on the tube wall with a marker pen.

6. Flatten one end of the copper wire, bend the flat part into a hook shape, wrap such a small bundle of copper wire with tin foil, and then sterilize.

7. Place the sterilized copper wire and anaerobic tube in the anaerobic workstation, dig out the small black spots with the bent end of the copper wire, and inoculate them in new medium. In order to ensure the purity of the bacteria distributed, during operation, small black spots should be dug out from the mouth of the tube to the inside of the tube one by one. Before each small black spot is dug out, a new sterilized copper wire should be replaced; After the black spot, dig out all the agar from the small black spot to the mouth of the tube.

8. Extract the DNA of the isolated target anaerobic bacteria. When PCR is used for 16s rDNA sequencing, no double peaks will appear, and the sequencing results are accurate. The comparison results in the NCBI database show that the bacteria is compatible with a Gram-positive strain. The warm bacteria are the most similar, with a similarity of 99%.

Example 2

1. Enrich 3 g of the Atlantic deep-sea hydrothermal vent bottom mud containing the target thalline with the liquid medium added with ferrous sulfate heptahydrate (0.0005g/L) indicator.

2. Under aerobic conditions, use ethanol (concentration of 10mL/L) as carbon source, ferrous sulfate heptahydrate (0.0005g/L) as indicator, and agar concentration at 0.03g/L, prepare medium 15mL, set In a Erlenmeyer flask with a cotton stopper, high temperature and high pressure sterilization (121° C., 15 minutes).

3. Before the culture medium is solidified, place the Erlenmeyer flask and the enrichment solution in the sample chamber of the anaerobic workstation, pump nitrogen and fill it with nitrogen repeatedly three times, and remove the air in the culture medium.

4. In the operation box of the anaerobic workstation, add 3mL of the enriched supernatant to the uncoagulated medium, shake well and distribute into anaerobic tubes.

5. Take out the anaerobic tube, put it into an ordinary incubator, and adjust the temperature to 60°C. After 35 hours, it can be observed that there are small black spots (color development phenomenon that reacts with the indicator) inside the medium, and continue to cultivate for 4 Every day, there will be small black spots of different sizes and different times, which are marked on the tube wall with a marker pen.

6. Flatten one end of the copper wire, bend the flat part into a hook shape, wrap such a small bundle of copper wire with tin foil, and then sterilize.

7. Place the sterilized copper wire and anaerobic tube in the anaerobic workstation, dig out the small black spots with the bent end of the copper wire, and inoculate them in new medium.

8. Extract the DNA of the isolated target anaerobic bacteria. When PCR is used for 16s rDNA sequencing, no double peaks will appear, and the sequencing results are accurate. The results in the NCBI database show that the bacteria is most similar to an anaerobic mesophilic bacteria , 100% similarity.

Example 3

1. Enrich 3-10mL of the surface water near the bottom of the Pacific Ocean containing the target bacteria with a liquid medium added with ferrous sulfate heptahydrate (0.02g/L) indicator.

2. Under aerobic conditions, sodium acetate (concentration is 8g/L) is used as carbon source, ferrous sulfate heptahydrate (0.02g/L) is used as indicator, and agar concentration is 0.03g/L, prepare medium 100mL, Place in a Erlenmeyer flask with a cotton stopper, and sterilize under high temperature and high pressure (121° C., 15 minutes).

3. Before the culture medium is solidified, place the Erlenmeyer flask and the enrichment solution in the sample chamber of the anaerobic workstation, pump nitrogen and fill it with nitrogen repeatedly three times, and remove the air in the culture medium.

4. In the operation box of the anaerobic workstation, add 3mL of the enriched supernatant to the uncoagulated medium, shake well and distribute into anaerobic tubes.

5. Take out the anaerobic tube, put it into an ordinary incubator, and adjust the temperature to 15°C. After 24 hours, small black spots can be observed inside the medium. Continue to cultivate for 2 days, there will be different sizes, and the time of appearance Different small black spots are marked on the tube wall with a marker pen.

6. Flatten one end of the copper wire, bend the flat part into a hook shape, wrap such a small bundle of copper wire with tin foil, and then sterilize.

7. Place the sterilized copper wire and anaerobic tube in the anaerobic workstation, dig out the small black spots with the bent end of the copper wire, and inoculate them in new medium.

8. Extract the DNA of the isolated target anaerobic bacteria. When PCR is used for 16s rDNA sequencing, no double peaks will appear, and the sequencing results are accurate. The results in the NCBI database show that the bacteria are most similar to Thioclava pacifica, with a similarity of 100%. . .

Claims (1)

1. a method for isolation and culture of anaerobic microorganisms, the isolation steps are: 1). Enrich 2.5 g of the bottom mud of the Pacific deep-sea hydrothermal vent containing the target bacteria with a liquid medium added with 0.0001 g/L of ferrous sulfate heptahydrate indicator; 2). Under aerobic conditions, use ethanol with a concentration of 0.2mL/L as a carbon source, 0.006g/L ferrous sulfate heptahydrate as an indicator, and an agar concentration of 1.8g/L to prepare 50mL of a culture medium. Sterilize in a Erlenmeyer flask with a cotton stopper at 121°C for 15 minutes under high temperature and high pressure; 3). Before the culture medium is solidified, place the Erlenmeyer flask and the enrichment solution in the sample chamber of the anaerobic workstation, pump nitrogen and fill it with nitrogen repeatedly three times, and remove the air in the culture medium; 4).In the operation box of the anaerobic workstation, add 3mL of the enriched supernatant to the uncoagulated medium, shake well, and distribute it into anaerobic tubes or transparent glass with well-tight screw plugs tube; 5). Take out the anaerobic tube, put it into an ordinary incubator, and adjust the temperature to 90°C. After 60 hours, small black spots can be observed inside the medium. Continue to cultivate for 2 days, and there will be different sizes, appearing successively Small black dots with different times are marked on the tube wall with a marker pen; 6). Flatten one end of the copper wire, bend the flat part into a hook shape, wrap such a small bundle of copper wire with tin foil, and then sterilize; 7). Place the sterilized copper wire and anaerobic tube in the anaerobic workstation, dig out the small black spots with the bent end of the copper wire, and inoculate them in a new medium; in order to ensure the Purity, during operation, dig out small black spots from the mouth of the tube to the inside of the tube one by one. Before digging out a small black spot, replace a new sterilized copper wire; Scoop out all the agar between this small black spot and the mouth of the tube.

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